Neutralization has been the most sensitive and widely used method for serotyping birnavirus isolates. Among most IPNV strains some crossneutralization has been observed, but three different groups have been established with the following reference strains: VR 299 for serotype 1, Ab for serotype 2 and Sp for serotype 3. A further strain N1 with a distinct antigenic structure has recently been isolated from Atlantic salmon in Norway. All aquatic birnaviruses crossreact serologically to some extent. A high degree of crossneutralization has been noted for OV and TV; fluorescent antibodies crossreacted with IPNV and TV.
For IBDV two definite serotypes could be established which show some residual crossneutralization. Serotype I encompasses the classical strains causing bursal disease in chicks, whereas serotype II differentiates the nonpathogenic strains originally isolated from turkeys.
Neutralizing antibodies raised against either serotype of IBDV precipitated VP2 in extracts prepared from infected CEC; these antibodies did not bind in immunoblots. It can be concluded, therefore, that the antigenic site responsible for the induction of neutralizing antibodies is highly conformation-dependent. The isolation of escape-mutants with neutralizing antibodies as selection barriers clearly showed that at least three independent types of epitope exist in the domain responsible for the induction of neutralizing antibodies. The structural basis for the formation of this conformation-dependent epitope is provided by two hydrophilic regions in the central part of the VP2 peptide. Exchange of one amino acid in one of the hypdrophilic parts is sufficient for altering the neutralizing specificity. Towards the C-terminal end of VP2, adjacent to this conformational site, identical amino acid sequences form a sequence-dependent epitope crossreacting with both serotypes. Considerable variation in the degree of neutralization has been noted among isolates in America, and vaccination failures have been attributed to these 'variants'. Such an antigenic drift could not be confirmed for strains isolated in Europe or in Africa.
Antigenic differences between serotypes I and II not only reside in VP2; structural protein VP3 can also be used for differentiating the two serotypes. Monoclonal antibodies could be prepared which were directed against VP3 of one serotype only, whereas other antibodies recognized a common epitope. The serotype-specific and the common epitopes are arranged on VP3 in a nonoverlapping manner. Formation of the common and type-specific epitopes is in agreement with identical and mismatching amino acid sequences yielding hydrophilic segments on the VP3 polypeptide. Amino acid exchanges clustered in hydrophobic parts did not interfere with the antigenic specificity. There is no serologic crossreactivity between IBDV and other birnaviruses.
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